Infectious pancreatic necrosis virus (IPNV) is an economically important fish pathogen. IPNV belongs to a group of viruses known as Birnaviridae (Brown (1986), Intervirology, 25:141–143). Other members of Birnaviridae include infectious bursal disease virus (IBDV) of fowl and Drosophila X virus. IPNV was discovered to be associated with a highly contagious disease of susceptible hatchery-reared trout and salmonoids. As the name indicates, the infection among trout produces marked pancreatic necrosis, but histopathological changes sometimes also occur in adjacent adipose tissue, in renal hematopoietic tissue, in the gut, and in the liver (Wolf et al. (1960), Proc. Soc. Exp. Biol. Med., 104:105–108). Histopathological changes can also occur in renal excretory and hematopoietic tissues, as first reported by Yasutake et al. (1965), Ann. N.Y. Acad. Sci., 126:520–530. Although renal damage is consistent with the high titer of virus typically found in kidneys, at least in carrier fish, focal degeneration of liver parenchymal cells in yearling Atlantic salmon which had been previously inoculated with IPNV was also noted (Swanson and Gillespie (1979), J. Fish. Res. Board Can., 36:587–591).
IPNV shows a high degree of antigenic heterogeneity. Three different stereotypes, Ab, Sp and VR299 (MacDonald and Gower (1981), Virology, 114:187–195; Okamoto et al. (1983), Eur. J. Fish Dis., 6:19–25) and ten subgroups (Heppell et al. (1992), J. Gen. Virol., 73:2863–2870) have been identified. IPNV is the etiological agent of a contagious, high mortality disease of young, hatchery-reared salmonids (Wolf et al. (1960), supra) and other non-salmonid fishes (Adair and Ferguson (1981), J. Fish Dis., 4:69–76). IPNV is a double-stranded RNA virus with four virion proteins (MacDonald and Dobos (1981), Virology, 114:414–422).
Birnaviruses possess a bisegmented (A and B), double-stranded RNA genome contained within a medium-sized, unenveloped, icosahedral capsid. Birnavirus gene expression involves the production of four unrelated major genes that undergo various post-translational cleavage to generate three to five structural proteins (Dobos, P. 1995. Annu. Rew. Fish Dis. 5, 25–54). The largest protein (90 kDa), VP1, is encoded by the smaller segment B genome, and the larger genome segment A encodes VP2 (42 kDa), VP4 (28 kDa) and VP3 (32 kDa). Genome segment A contains an additional small open reading frame (ORF) which overlaps the amino terminal end of the polyprotein from the reading frame (Duncan, R., Nagy, E., Krell, P. J. and Dobos, P. 1987. J. Virol. 61, 3655–3664). This small ORF encodes a 17 kDa arginine-rich minor polypeptide, VP5, which is produced in small quantities and is synthesized during the early replication cycle (Magyar, G. & Dobos, P. 1994. Virology 204, 580–589).
There are two major morphologically and biochemically distinct modes of death in eukaryotic cells: apoptosis and necrosis (Duvall and Wyllie (1986), Immunol. Today, 7:115–119). Apoptosis is a physiological process involved in normal tissue turnover that occurs during embryogenesis, aging, and tumor regression, but pathological stimuli, such as viral infections (Gougeon and Montagnier (1993), Science, 260:1269–1270), can also be triggering factors. Typically, apoptotic cell death is characterized by nuclear condensation, endonucleolytic degradation of DNA at nucleosomal intervals (“laddering”), and plasma membrane blebbing (Wyllie et al. (1980), Int. Rev. Cytol., 68:251–306). Necrosis is a pathological reaction that occurs in response to perturbations in the cellular environment such as complement attack, severe hypoxia, or hyperthermia. These stimuli increase the permeability of the plasma membrane, resulting in irreversible swelling of the cells (Wyllie et al,(1980), supra).
Apoptosis is important during embryonic development, metamorphosis, tissue renewal, hormone-induced tissue atrophy and many pathological conditions. In multi-cellular organisms, apoptosis ensures the elimination of superfluous cells including those that are generated in excess, have already completed their specific functions or are harmful to the whole organism. In reproductive tissues, massive cell death occurs under the control of hormonal signals. A growing body of evidence suggests that the intracellular “death program” activated during apoptosis is similar in different cell types and conserved during evolution. (Hengartner and Horvitz (1994), Cell, 76:1107–1114). In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing the emergence of cancer.
Apoptosis involves two essential steps. The “decision” step is controlled by the Bcl-2 family of proteins which consists of different anti- and pro-apoptotic members. The “execution” phase of apoptosis is mediated by the activation of caspases and cysteine proteases that induce cell death via the proteolytic cleavage of substrates vital for cellular homeostasis.
Bcl-2 protein is a 25 kD integral membrane protein of the mitochondria. Bcl-2 protein extends survival in many different cell types by inhibiting apoptosis elicited by a variety of death-inducing stimuli (Korsmeyer (1992), Blood, 80:879–886). Overexpression of bcl-2 has been related to hyperplasia, autoimmunity and resistance to apoptosis (Fang et al. (1994), J. Immunol., 153:4388–4398). Bcl-2 contains a family of related genes, which includes, but is not limited to, A1, mcl-1, bcl-w, bax, bad, bak and bcl-x. A1, mcl-1, bcl-2 and bcl-x1 (long form of bcl-x) are presently known to confer protection against apoptosis and are referred to herein as “anti-apoptotic bcl-2 related proteins”. In contrast, bax, bad, bak and bcl-xs (short form of bcl-x) are presently known to promote cell death by inhibiting this protective effect.
Mcl-1 is one of the members of the Bcl-2 family. Like Bcl-2, Mcl-1 heterodimerizes with Bax, an accelerator of apoptosis in the Bcl-2 family, and neutralizes the cytotoxicity induced by Bax in yeast (Bodrug et al. (1996), Death Differ., 2:173–182). Mcl-1 is also able to protect Chinese hamster ovary cells from apoptosis induced by c-myc overexpression (Reynolds et al. (1994), Cancer Res., 54:6348–6352). This protein was discovered as a novel gene induced early in the induction of differentiation of a human myeloid leukemia cell line (Kozopas et al. (1993), Proc. Natl. Acad. Sci. USA, 90:3516–3520). Expression of Mcl-1 mRNA was rapidly up-regulated with phorbol ester in those cells followed by a rapid degradation, consistent with the presence of a mRNA destabilization sequence in its 3′-untranslated region. The half-life of the Mcl-1 protein is short (Yang et al. (1995), J. Cell Biol., 128:1173–1184), which has been ascribed to the presence of two PEST (proline, glutamic acid, serine, threonine) motifs. Therefore, Mcl-1 is suggested as a rapidly inducible, short-term effector of cell viability (Yang et al. (1996), J. Cell. Physiol., 166:523–536). Recently, Hong et al. (Virology, (1998), 250:76–84) reported that an E1-S of IPNV Ab strain induced apoptosis in CHSE-214 cells. Hong et al.'s publication is herein incorporated by reference. In Hong et al.'s report, four kinds of detecting methods were used to determine whether apoptosis is involved in fish embryonic cell death after IPNV infection: (1) assay with terminal deoxynucleotidyl transferase (TdT)-mediated end-labeling of DNA in nuclei of intact cells during virus infection; (2) assay for procoagulant activity; (3) assay for DNA ladders; and (4) electron microscopic assays for the ultrastructural changes in characteristic apoptotic cells.
The results show that apoptosis precedes any detectable necrotic change in CHSE-214 cells, suggesting that apoptosis characterizes the onset of pathology in host cells and is followed by necrotic processes. Hong et al.'s report is important because previously, IPNV infection is only viewed as caused by a necrotic process. However, Hong et al.'s report did not provide any insights which delineate the apoptotic process from necrosis.
In the present invention, an investigation of apoptosis is carried out by using CHSE-214 cells infected with IPNV as a model. The investigation is conducted by transfecting the cells with a pEGFP vector which enables the cells to express EGFP (a variant type of GFP [green fluorescent protein]). Based on the special characteristics of EGFP which can fluorescence 35 times more intense than the wild-type GFP, the morphological changes during apoptosis are monitored, which show that IPNV causes CHSE-214-EGFP cells to undergo apoptosis, then a nontypical apoptosis, and finally, postapoptotic necrosis in cells. The discovery of the nontypical apoptosis stage before necrosis takes place is one of the novel findings in the present invention.
The present invention also provides studies of apoptosis via an Mcl-1 dependent pathway. The results of the present invention indicate that the occurrence of apoptosis is due to down regulation of the Mcl-1 gene caused by viral infection. In addition, various drugs or chemicals are tested for their capacity of preventing the down-regulation of Mcl-1 protein expression by viral infection. The results show that by blocking the down regulation of the Mcl-1 gene, the cell death caused by IPNV infection is effectively prevented.
The present invention is important because it not only provides a model for studies of apoptosis but also provides a means for preventing or containing widespread of IPNV infection in aquatic organisms.